NEGATIVE REGULATORY ROLE OF PI3-KINASE IN TNF-INDUCED TUMOR
Susanne MATSCHURAT1, Sabine BLUM1, Rita MITNACHT-KRAUS1, Henry B.P.M. DIJKMAN2, Levent KANAL1, Robert M.W. DE WAAL2
and Matthias CLAUSS1*
1Department of Molecular Cell Biology, Max-Planck-Institute for Physiological and Clinical Research, Bad Nauheim, Germany
2Department of Pathology, University Medical Centre Nijmegen, HB Nijmegen, The Netherlands
Tissue factor is the prime initiator of blood coagulation.
Expression of tissue factor in tumor endothelial cells leads to
thrombus formation, occlusion of vessels and development of
hemorrhagic infarctions in the tumor tissue, often followed
by regression of the tumor. Tumor cells produce endogenous
vascular endothelial growth factor (VEGF), which sensitizes
endothelial cells for systemically administered tumor necro-
sis factor ? (TNF ?) and synergistically enhances the TNF-
induced expression of tissue factor. We have analyzed the
pathways involved in the induction of tissue factor in human
umbilical cord vein endothelial cells (HUVECs) after com-
bined stimulation with TNF and VEGF. By using specific low
molecular weight inhibitors, we demonstrated that protein
kinase C (PKC), p44/42 and p38 mitogen-activated protein
(MAP) kinases, and stress-activated protein kinase (JNK) are
essentially involved in the induction of tissue factor. In con-
trast, the application of wortmannin, an inhibitor of phospha-
tidylinositol 3 (PI3)-kinase, led to strongly enhanced expres-
sion of tissue factor in TNF- and VEGF-treated cells,
implicating a negative regulatory role for PI3-kinase. In vivo,
the application of wortmannin promoted the formation of
TNF-induced hemorrhages and intratumoral necroses in mu-
rine meth A tumors. The co-injection of wortmannin low-
ered the effective dose of applied TNF. Therefore, it is con-
ceivable that the treatment of TNF-sensitive tumors with a
combination of TNF and wortmannin will ensure the selec-
tive damage of the tumor endothelium and minimize the risk
of systemic toxicity of TNF. TNF-treatment in combination
with specific inhibition of PI3-kinase is a novel concept in
© 2003 Wiley-Liss, Inc.
Key words: tumor necrosis factor; PI3-kinase; tissue factor; vascular
targeting; anti-tumor therapy
Vascular targeting, a strategy for cancer treatment, aimed at
activated endothelial cells, offers several advantages over targeting
tumor cells directly. Compared to tumor cells, endothelial cells
constitute a genetically stable, diploid and fairly homogenous
population, which is unlikely to acquire mutations that might
render them insensitive to therapy. Furthermore, endothelial cells
are in direct contact to the blood stream and therefore can be easily
reached by systemic therapy. Because any single tumor vessel
supplies a large number of tumor cells with oxygen and nutrients,
the targeted disruption of relatively few vessels can cause the death
of a multitude of tumor cells.1–3
A potent agent for activating tumor endothelial cells is tumor
necrosis factor ? (TNF ?). TNF was discovered based on its ability
to induce tumor necrosis in the murine meth A fibrosarcoma.4
Although originally believed to act directly on tumor cells, later
studies revealed that systemic injection of a low and nontoxic dose
of TNF into mice bearing meth A fibrosarcomas leads to the
thrombotic occlusion of tumor vessels.5–8Other types of tumors
that are relatively resistant to TNF-treatment have been shown to
respond to TNF in combination therapy, e.g., with interferon-?.9
The deposition of fibrin clots in tumor vessels could be demon-
strated to be dependent on tissue factor, which is the major initiator
of blood coagulation.10Targeted expression of tissue factor in
tumor blood vessels causes tumor necrosis and subsequent regres-
sion of tumors, therefore constituting a promising concept of
anti-tumor therapy.11–13TNF selectively affects neovasculature,
including tumor blood vessels, without activating quiescent endo-
thelial cells elsewhere in the body. Therefore, it was postulated,14
and subsequently shown, that tumor cells produce factors that
prime the tumor endothelium for the action of TNF,15–17e.g.,
VEGF.15Although VEGF is primarily known as a major player in
angiogenesis and vasculogenesis,18it also initiates endothelial
procoagulant activity and vascular permeability.15,19These activ-
ities have been suggested to contribute to the sensitization of the
tumor vasculature and cause tumors to undergo TNF-mediated
hemorrhagic necrosis.7,14,20Of note, tumor endothelial cells are the
only endothelium expressing tissue factor in vivo, to a lower
degree without treatment and strongly enhanced after systemic
infusion of TNF.10,21Endothelial cells of tissues other than tumors
(an exception is the spleen) do not express tissue factor, even after
treatment with lethal concentrations of endotoxin.22
Tissue factor expression in endothelial cells in vitro can be
induced by several cytokines, including TNF and interleukin-1,
and by VEGF.23,24In addition, it was shown that VEGF synergis-
tically enhances the TNF-induced expression of tissue factor.15
TNF-mediated endothelial tissue factor expression could be linked
to the activation of the transcription factors NF?B and Egr-1.25–27
Whereas NF?B is not involved in VEGF-induced endothelial
tissue factor expression, Egr-1 plays an essential role.27,28The
induction of tissue factor expression in human endothelial cells
after costimulation with TNF and VEGF is mediated via binding of
the factors to TNFR-1 and VEGFR-2 (KDR), respectively.29,30
Autophosphorylation of the activated VEGFR-2 leads to the re-
cruitment and activation of phospholipase C?. Phospholipase C?
Abbreviations: HUVECs, human umbilical cord vein endothelial cells;
JNK, stress-activated protein kinase; MAP kinase, mitogen-activated pro-
tein kinase; meth A, methylcholanthrene A; PI3-kinase, phosphatidylino-
sitol 3-kinase; PKC, protein kinase C; TBS, TRIS-buffered saline; DMSO,
dimethylsulfoxide TNF ?, tumor necrosis factor ?; VEGF, vascular endo-
thelial growth factor.
Grant Sponsor: European Community (EC); Grant number: BMH4-
Sabine Blum’s current address is: U3pharma, 82152 Munich, Germany.
Dr. Clauss’s current address is: Indiana Center for Vascular Biology and
Medicine, Indiana School of Medicine, Indianapolis, IN 46202, USA.
*Correspondence to: Department of Cellular and Integrative Physiology,
Indiana Center for Vascular Biology and Medicine, 975 West Walnut
Street, IB 433, Indianapolis, IN 46202 USA. Fax: ?1-317-278-0089.
Received 28 February 2003; Revised 13 May 2003; Accepted 15 May
Int. J. Cancer: 107, 30–37 (2003)
© 2003 Wiley-Liss, Inc.
Publication of the International Union Against Cancer
thereby generating diacylglycerol, which is the main activator of
protein kinase C (PKC). PKC activation precedes the activation of
the Raf/MEK/p44/42 MAP kinase signaling cascade and is in-
volved in VEGF-mediated tissue factor induction,27and also in
VEGF-mediated proliferation and vascular permeability.31,32
P44/42 MAP kinase, p38 MAP kinase and p54/46 JNK belong to
the family of mitogen- and stress-activated protein kinases. These
kinases become activated by growth factors, cytokines and stress-
induced mediators.33,34In addition to p44/42 MAP kinase, VEGF
also stimulates the p38 MAP kinase signaling pathway, mediating
endothelial cell migration35and tissue factor production.36TNF
activates all the 3 above mentioned kinases, p44/42 MAP kinase,
p38 MAP kinase and JNK. Like VEGF, TNF induces the expres-
sion of tissue factor via the p44/42 MAP kinase signaling path-
way.27A further signaling enzyme activated in response to TNF-
or VEGF-treatment is phosphatidylinositol 3-kinase (PI3-ki-
nase).37–42Activated PI3-kinase converts membrane phospholip-
ids into phosphatidylinositol triphosphate, which in turn binds
protein kinase Akt.43Recently we showed that inhibition of PI3-
kinase by its specific inhibitor wortmannin leads to synergistically
enhanced expression of tissue factor in VEGF-stimulated endothe-
lial cells. Furthermore, expression of a constitutively active Akt-
mutant led to a reduced expression of tissue factor protein in
response to VEGF.36
In the present study, signaling pathways likely to be involved in
the synergistic induction of tissue factor in HUVECs by TNF and
VEGF were analyzed. Inhibition of either PKC, p44/42 MAP
kinase, p38 MAP kinase or JNK by small chemical compounds led
to a partial abrogation of TNF- and VEGF-induced tissue factor
production, indicative of their contribution to the synergism ob-
served. In striking contrast, inhibition of the PI3-kinase signaling
pathway by wortmannin further enhanced the tissue factor produc-
tion induced by TNF and VEGF, indicating that PI3-kinase is a
negative regulator of TNF- and VEGF-induced tissue factor pro-
duction in vitro. In vivo, wortmannin clearly promoted the TNF-
mediated development of intratumoral hemorrhages and necroses
in meth A tumors. Because the co-application of wortmannin
reduced the effective dose of administered TNF, inhibition of the
PI3-kinase can be considered a strategy to minimize toxic side
effects of a TNF-based anti-tumor therapy.
MATERIAL AND METHODS
Tissue culture media and supplements were obtained from
Gibco Life Technologies (Eggenstein, Germany). Human umbili-
cal cords were kind donations from local hospitals. Citrated plasma
was obtained from whole blood samples. VEGF165 was expressed
in Pichia pastoris and purified in our laboratory according to a
protocol from Invitrogen (Karlsruhe, Germany). Recombinant hu-
man TNF ? for in vitro experiments was obtained from Cell
Concepts (Umkirch, Germany), whereas recombinant human TNF
? for in vivo experiments was kindly donated by D. Ma ¨nnel
(Regensburg, Germany). The TNF used for in vivo experiments
was produced for use in patients and is therefore essentially
endotoxin-free. PD98059, SB203580, Bim I and wortmannin were
purchased from Calbiochem (Bad Soden, Germany). CEP-11004
was obtained from Cephalon, Inc. (West Chester, PA). All further
reagents were purchased from Sigma Chemical Co. (Munich,
Germany) unless otherwise indicated.
Human umbilical cord vein endothelial cells (HUVECs) were
prepared as previously described.44Cells were cultured in
MCDB131 medium that was supplemented with 8% fetal bovine
serum, 2% human serum, 4 mM L-glutamine, 100 units/ml peni-
cillin, 100 ?g/ml streptomycin, 2.5 ?g/ml amphotericin B, 10
?g/ml heparin and 0.4% endothelial cell growth supplement (Pro-
mocell, Heidelberg, Germany). Confluent cells plated on 6-well
plates were used for all experiments.
Tissue factor activity determination
Tissue factor expression was determined by a 1-stage clotting
assay. Cells were incubated with 500 pM TNF and/or 1 ng/ml
VEGF in MCDB131 medium that was supplemented with 8% fetal
bovine serum, 4 mM L-glutamine, 100 units/ml penicillin and 100
?g/ml streptomycin in the presence or absence of small peptide
inhibitors for 4 hr. Inhibitor concentrations were 5 ?M of Bim I,
10 ?M of both PD98059 and SB203580, 500 nM of CEP-11004
and 100 nM of wortmannin, also in the FACS and Western blot
experiments. Assays were performed with whole cells obtained in
suspension after scraping off the dish with a rubber policeman.
Tissue factor equivalents were determined as previously de-
scribed.15In brief, 100 ?l of the resuspended cells were mixed
with 100 ?l of citrated plasma, and after recalcification with 100
?l of a 25 mM CaCl2solution, clotting times were measured. A
standard curve of purified human tissue factor was used to deter-
mine tissue factor equivalents.
To determine tissue factor expression by cytofluorometric anal-
ysis, confluent HUVECs were stimulated with 500 pM TNF and/or
1 ng/ml VEGF in MCDB131 medium supplemented with 8% fetal
bovine serum, 4 mM L-glutamine, 100 units/ml penicillin and 100
?g/ml streptomycin in the presence or absence of small peptide
inhibitors for 6 3/4 hr. Cells were detached with 5 mM EDTA/25
mM HEPES in Hank’s Balanced Salt Solution. Incubation with the
primary unconjugated tissue factor monoclonal antibody TF9-
6B445(kind donation from W. Ruf, LaJolla, CA) at a final con-
centration of 20 ?g/ml was carried out for 30 min at 4°C. After
washing off unbound antibody with FACS-buffer (2.5% fetal
bovine serum/0.1% NaN3in PBS), the cells were incubated with
the secondary phosphatidylethanolamine-conjugated goat anti-
mouse antibody (Dianova, Hamburg, Germany) for 20 min at 4°C.
Flow cytometry analysis was performed with a FACScan (Becton-
Dickinson, Heidelberg, Germany) using the software program
Cellquest. The mouse IgG MOPC-21 (Sigma Chemical Co., De-
isenhofen, Germany) was used as an isotype-control to determine
unspecific background staining. An antibody directed against E-
Selectin (Pharmingen, Hamburg, Germany) was used as a marker
for endothelial cells.
Western blot analysis
For Western blot analysis HUVECs were starved in MCDB131
medium supplemented with 8% fetal bovine serum, 4 mM L-
glutamine, 100 units/ml penicillin and 100 ?g/ml streptomycin for
4 hr. Where indicated, 100 nM wortmannin was added to the
medium 1 hr before stimulation of the cells with 500 pM recom-
binant human TNF ? for 20 min and/or 1 ng/ml recombinant
VEGF165 for 5 min. After stimulation, the cells were washed with
PBS and subsequently lysed by adding 150 ?l Laemmli buffer/
well. The samples were boiled at 95°C for 10 min and loaded onto
15% SDS/PAGE gels. Proteins were separated by electrophoresis
and blotted onto nitrocellulose (Schleicher & Schu ¨ll, Dassel, Ger-
many) using a semidry blotting apparatus. Unspecific binding was
reduced by blocking the membrane in Tris-buffered saline (TBS)/
0.1% Tween 20/5% nonfat dry milk. The membranes were incu-
bated with primary antibodies directed against phospho-p44/42
MAP kinase, phospho-p38 MAP kinase, phospho-JNK or p44/42
MAP kinase (Cellsignaling, Frankfurt, Germany) according to the
manufacturer’s instructions. Blots were developed using an en-
hanced chemiluminescence kit (Amersham Pharmacia Biotech,
Freiburg, Germany). To reprobe the same membrane with a dif-
ferent antibody, bound antibodies were stripped off using Pierce
Restore Western Blot Stripping Buffer (Pierce, Sankt Augustin,
Germany). Blocking of the membrane was repeated and another
antibody was applied.
In vivo tumor growth
The animal study protocols were performed according to the
permission of the Regierungspra ¨sidium Darmstadt (II25.3.-19c20/
PI3-KINASE IN TNF-INDUCED TUMOR NECROSIS
15-B2/110). Methylcholanthrene A-transformed murine fibrosar-
coma cells (BFS-1 cells), raised on the C57black/6 background,
were chosen for in vivo tumor studies. To inoculate tumors, 1.5 ?
106BFS-1 cells resuspended in PBS were injected subcutaneously
into the back of C57Bl/6 mice. On day 10 after inoculation, solvent
(11 mg DMSO), 0.4 mg/kg body weight wortmannin, 5 ?g TNF,
or 1 ?g TNF ? 0.4 mg/kg body weight wortmannin were injected
intraperitoneally. At the time-points indicated (either 3 or 24 hr
after injection), mice were sacrificed and tumors were harvested.
The tumors were embedded in Tissue Tek medium (Sakura,
Zoeterwoude, The Netherlands), snap-frozen, and stored at
Calculation of necrotic areas and statistical analysis
To evaluate intratumoral necrosis formation in response to treat-
ment with TNF and/or wortmannin for 24 hr, morphometric anal-
ysis was performed. Multiple cryosections (5 ?m) of each tumor
were stained with hematoxylin/eosin to determine tissue morphol-
ogy. The extent of necrotic areas within the tumors was calculated
as percentage of total tumor area and the results are depicted with
a box-blot in a “Box-Blot”-diagram. Statistical evaluation was
performed using the Mann-Whitney Rank Sum Test. p ? 0.05 was
Cryosections (5 ?m) of meth A fibrosarcomas were double-
stained with an antibody that recognizes fibrin and hybridoma
supernatant specific for CD31 to selectively detect blood vessels.
Sections were fixed in 100% acetone for 10 min at room temper-
ature. Thereafter, sections were incubated with FITC-conjugated
polyclonal goat anti-mouse fibrinogen antibody (Nordic Immuno-
logical Laboratories, Tilburg, The Netherlands) that was diluted
1:100 in dilution buffer (Immustain, Llanberies, UK) for 45 min at
room temperature. After extensive washing in PBS, sections were
incubated in Tris-buffered saline (TBS)/1% bovine serum albumin
(BSA)/5% goat serum for 30 min to block unspecific binding
epitopes. Consecutively, sections were incubated with CD31-spe-
cific hybridoma supernatant Mec 13.3 (ATCC, Manassas, VA) for
2 hr at room temperature. Unbound antibody was removed by
washing in TBS followed by 2 washing steps in TBS containing
0.1% Tween-20. Indocarbocyanin (Cy 3)-conjugated goat anti-rat
IgG secondary antibody (Dianova, Hamburg, Germany), diluted
1:1,000 in TBS/1% BSA, was applied to the sections for 1 hr at
room temperature. After 3 final washing steps, the sections were
mounted in Mowiol (Calbiochem, Bad Soden, Germany) and
stored in the dark at 4°C.
Protein kinase C, p44/42 and p38 MAP kinases, and JNK
mediate the synergistic expression of tissue factor by TNF and
Both TNF and VEGF induce the expression of tissue factor
protein in human umbilical vein endothelial cells (HUVECs), and
combined stimulation with both factors leads to a synergistic effect
on procoagulant activity (Fig. 1 and Reference 15). We and others
have previously shown that protein kinase C (PKC), and both
p44/42 and p38 MAP kinases, are critically involved in the induc-
tion of tissue factor protein after stimulation of HUVECs with
VEGF.27,36The role of these 3 kinases in the synergistic induction
of tissue factor by TNF and VEGF was evaluated in the present
experiments by using low molecular weight inhibitors known to
interfere with these signaling pathways. Treatment of HUVECs
with 5 ?M of the PKC-specific inhibitor Bisindolylmaleimid I
(Bim I), which interacts with the ATP-binding site of the catalytic
domain of members of the PKC-family, reduced TNF- and VEGF-
induced tissue factor activity by ?74% (Fig. 1a). Furthermore,
addition of PD98059, a p44/42 MAP kinase pathway inhibitor, led
to a reduction of tissue factor expression of ?50% in TNF- and
VEGF-stimulated cells (Fig. 1b). Likewise, the application of the
p38 MAP kinase-specific inhibitor SB203580, which competes for
the ATP-binding site of the kinase, reduced the TNF- and VEGF-
mediated expression of tissue factor in HUVECs by ?73% (Fig.
1b). The combined treatment of TNF- and VEGF-stimulated cells
with inhibitors PD98059 and SB203580 resulted in a reduction of
tissue factor expression of ?92% (Fig. 1b). Similar results were
FIGURE 1 – Tissue factor-expression in HUVECs after stimulation
with TNF and VEGF in the presence or absence of specific inhibitors.
HUVECs were stimulated with 1 nM VEGF, 500 pM TNF or with
both factors for 4 hr in the presence/absence of 5 ?M Bim I (a), 10 ?M
PD98059 and/or 10 ?M 203580 (b) or 500 nM CEP-11004 (c).
Control cells were incubated in medium only. Tissue factor-expression
was assessed in a one-stage coagulation assay as described in Material
and Methods. The mean ? SD of double determinations is shown. Of
note, in some cases the error bars are too small to be visible. Exper-
iments were repeated at least 4 times.
MATSCHURAT ET AL.
obtained if 2 other inhibitors of p44/42 and p38 MAP kinases,
PD184352 and SB202190, respectively, were applied (data not
shown). Besides activation of the p44/42 and p38 MAP kinases, a
strong involvement of the stress-activated protein kinase JNK in
TNF- and VEGF-induced tissue factor expression could be dem-
onstrated. Application of the JNK-specific inhibitor CEP-11004
led to a reduction of tissue factor expression in HUVECs by ?60%
(Fig. 1c). These results indicate that PKC, p44/42 and p38 MAP
kinases, and JNK, are critically involved in the regulation of the
synergistic induction of tissue factor expression by TNF and
VEGF in HUVECs.
Negative regulatory role of PI3-kinase in TNF- and
VEGF-mediated tissue factor expression in HUVECs
Based on our recent identification of the PI3-kinase as a nega-
tive regulator of VEGF-mediated tissue factor expression in
HUVECs,36the role of PI3-kinase in the synergistic induction of
tissue factor by TNF and VEGF was analyzed. For this purpose,
wortmannin, an inhibitor specific for the PI3-kinase signaling
pathway, was added to the HUVEC-medium together with TNF
and VEGF. As demonstrated in Figure 2a, application of 100 nM
wortmannin led to a ?3-fold increase in tissue factor activity in
TNF- and VEGF-stimulated cells. Stimulation of HUVECs with
either factor alone revealed that both VEGF- and TNF-mediated
tissue factor expression were enhanced by wortmannin. However,
the procoagulant effect of wortmannin was more pronounced on
TNF-stimulated HUVECs than on VEGF-stimulated cells. Wort-
mannin only induced a minor increase in procoagulant activity in
HUVECs compared to untreated control cells.
The increased surface expression of tissue factor induced by
TNF and VEGF in combination with wortmannin in HUVECs was
confirmed by flow-assisted cytofluorometric analysis (FACS) us-
ing a monoclonal anti-tissue factor antibody (Fig. 2b). Like in the
clotting experiment (Fig. 2a), TNF was more potent in inducing
tissue factor expression in HUVECs than VEGF (Fig. 2b, upper
panel). Combined stimulation of the cells with TNF and VEGF
resulted in synergistically enhanced expression of tissue factor
(Fig. 2b, upper panel). Furthermore, TNF- and VEGF-mediated
tissue factor expression was further synergistically enhanced by
application of wortmannin (Fig. 2b, lower panel). In addition to the
representative FACS-analysis experiment shown in the 2 panels in
Fig. 2b, mean tissue factor expression in stimulated vs. nonstimu-
lated HUVECs calculated from several experiments is depicted in
Wortmannin enhances the activation of mitogen- and
stress-activated protein kinases
After having shown that the activation of p44/42 MAP kinase,
p38 MAP kinase and JNK is essential for the induction of tissue
factor in TNF- and VEGF-stimulated HUVECs (Fig. 1), we in-
vestigated whether PI3-kinase might have an inhibitory influence
on either of these signaling pathways. This could in part explain
the observed synergistic induction of tissue factor in the presence
of wortmannin. Cell lysates of HUVECs stimulated with TNF,
VEGF and wortmannin were subjected to Western blot analysis.
The activation of the respective kinases was determined by prob-
ing the membranes with specific antibodies directed against the
phosphorylated form of p44/42 MAP kinase, p38 MAP kinase and
JNK, respectively (Fig. 3). A pan-specific antibody for p44/42
MAP kinase was used to evaluate overall protein loading. Whereas
stimulation of the HUVECs with VEGF led to a stronger activation
of p44/42 MAP kinase than stimulation with TNF (upper panel),
TNF was a more potent activator for both p38 MAP kinase and
JNK than VEGF (middle panels). In either case, stimulation of
cells with both TNF and VEGF further enhanced the phosphory-
lation intensity of the respective kinases. If wortmannin was added
to the TNF- and VEGF-treated cells, the phosphoryation levels of
the p44/42 and the p38 kinases were indeed slightly increased.
However, the most dramatic increase in phosphorylation intensity
was observed for JNK. Of note, treatment of HUVECs only with
wortmannin did not lead to an increased phosphorylation intensity
of any of the kinases analyzed. This indicates that inhibition of the
PI3-kinase indeed leads to enhanced phosphorylation of the mito-
gen- and stress-activated kinases by TNF and VEGF.
Effect of wortmannin on tumor blood vessels in vivo
Based on our observation that TNF, VEGF and wortmannin
exhibit a strong synergistic effect on the induction of tissue factor
expression in HUVECs, we investigated the procoagulant effect of
wortmannin in combination with TNF on tumor vessels in vivo.
FIGURE 2 – Effect of the PI3-kinase-specific inhibitor wortmannin
on the tissue factor expression in HUVECs. (a) Cells were stimulated
with 1 nM VEGF, 500 pM TNF or with both factors. Wortmannin (100
nM) was added where indicated. Control cells were cultured in me-
dium only. Expression of tissue factor was determined by measure-
ment of procoagulant activity. Shown are the mean ? SD of double
determinations of a representative experiment. (b) Tissue factor-ex-
pression assessed by FACS-analysis as described in Material and
Methods using the tissue factor-specific monoclonal antibody TF9-
PI3-KINASE IN TNF-INDUCED TUMOR NECROSIS
Murine meth A fibrosarcomas were chosen as a model, because
previously they were shown to produce high endogenous levels of
VEGF.15Meth A tumor-bearing mice were either treated with
solvent only, 0.4 mg/kg bodyweight wortmannin, 1 ?g TNF, 5 ?g
TNF or a combination of 1 ?g TNF and 0.4 mg/kg bodyweight
wortmannin (Fig. 4). After 24 hr intratumoral bleedings were
observed in the mice treated with 5 ?g TNF (Fig. 4e, arrows).
However, the bleedings were more pronounced in tumors of mice
that had received the lower dose of 1 ?g TNF in combination with
wortmannin (Fig. 4g, arrows). In contrast, the tumors of mice
treated with 1 ?g TNF hardly exhibited any bleedings (data not
shown). Control tumors of mice treated with solvent (Fig. 4a) or
wortmannin (Fig. 4c) did not display any bleedings. Hematoxylin/
eosin-staining of representative cryosections revealed extensive
intratumoral necrosis formation in the mice treated with 1 ?g
TNF ? wortmannin (Fig. 4h, arrowheads), which was less prom-
inent after treatment with 5 ?g TNF (Fig. 4f, arrowheads) and even
lower after treatment with 1 ?g TNF (see Fig. 4i). No necrosis
formation was observed in the control tumors treated with solvent
or wortmannin (Fig. 4b?d). Morphometric analysis indicates a
significant increase in intratumoral necrosis formation in mice
treated with 1 ?g TNF ? wortmannin vs. treatment with either 1
?g TNF (p?0.003; Mann-Whitney Rank Sum Test) or wortman-
nin only (p?0.002) (Fig. 4i). Necrosis formation was rather con-
fined to the central portions of the tumors. The peripheral areas of
the tumors were hardly affected.
Tissue factor is the prime physiological initiator of the coagu-
lation cascade. In confirmation of previous studies showing that
the increased expression of tissue factor indeed leads to intravas-
cular fibrin formation,10occluded tumor vessels were detected
after 3 hr of treatment with 1 ?g TNF ? wortmannin. This was
shown by staining tumor sections with a FITC-labeled antibody
recognizing fibrin (Fig. 4m, arrows). In contrast, tumors of control
mice, treated with solvent only, lacked such occluded vessels (Fig.
4k). Double-staining of the respective sections with an endothelial
cell-specific antibody (?-CD31) confirmed that the stained struc-
tures were blood vessels (Fig. 4l?n).
These data indicate that wortmannin indeed enhances the pro-
coagulant activity of tumor blood vessels in vivo in TNF-treated
mice and promotes the development of intratumoral hemorrhages
In the present study, we showed that combined stimulation of
endothelial cells with TNF and VEGF induces several signal
transduction pathways that either promote or inhibit endothelial
tissue factor production. Using specific chemical inhibitors, pro-
tein kinase C, the mitogen-activated protein kinases p44/42 and
p38 and the stress-activated protein kinase JNK were identified as
positive regulators of tissue factor expression (Fig. 1). In contrast,
the PI3-kinase signaling pathway negatively regulates endothelial
tissue factor expression, as was shown by the application of the
PI3-kinase-specific inhibitor wortmannin (Fig. 2 and Shen et al.30).
A negative regulation of the mitogen- and stress-activated pro-
tein kinases by PI3-kinase could be responsible for the enhanced
expression of tissue factor in HUVECs stimulated with TNF and
VEGF in the presence of the chemical inhibitor wortmannin. This
potential mechanism is based on the observation that inhibition of
PI3-kinase by wortmannin resulted in a slightly increased phos-
phorylation intensity of p44/42 MAP kinase and p38 MAP kinase,
and a very prominent increase in JNK phosphorylation (Fig. 3).
PI3-kinase/Akt is known to negatively regulate the p44/42 MAP
kinase pathway.46,47This interaction was shown to be of impor-
tance for cell survival of HUVECs stimulated with TNF42and also
in VEGF-mediated tissue factor production.36In addition, inhibi-
tion of PI3-kinase in VEGF-stimulated endothelial cells was
shown to result in enhanced p38 MAP kinase activity leading to an
increase in tissue factor expression36and a higher apoptosis rate.48
In the present study, we observed that phosphorylation of JNK was
even stronger than phosphorylation of p44/42 MAP kinase and p38
MAP kinase in HUVECs in response to treatment with TNF ?
VEGF ? wortmannin. These results are in line with previously
published data from Madge and Pober,42who used LY294002 to
specifically inhibit PI3-kinase. Read et al.49were able to link the
activation of JNK and p38 MAP kinase to the activation of the
transcription factor AP-1 in TNF-induced expression of E-selectin.
Since the promoter sequence of the tissue factor gene contains 2
consensus sites for AP-1 binding, and AP-1 was identified as a
positive regulator of tissue factor expression in endothelial cells
stimulated with TNF,25enhanced phosphorylation of JNK in
HUVECs treated with TNF ? VEGF ? wortmannin could like-
wise result in activation of AP-1. In addition, both TNF- and
VEGF-induced tissue factor expression depends on the activation
of the transcription factor Egr-1 mediated by the p44/42 MAP
kinase signaling pathway.27Taken together, these data strongly
argue for a link between PI3-kinase, mitogen- and stress-activated
protein kinases, and the transcription factors AP-1 and Egr-1 in the
regulation of tissue factor production.
It should be taken into consideration that the PI3-kinase/Akt
signaling-pathway is also suggested to be of importance for the
survival of cells, at least in an anchorage-dependent manner.50
TABLE I – MEAN TISSUE FACTOR EXPRESSION IN HUVECS
DETERMINED BY FACS-ANALYSIS1
TNF ? VEGF
VEGF ? wortmannin
TNF ? wortmannin
TNF ? VEGF ? wortmannin
0.40 ? 0.26
0.64 ? 0.51
1.76 ? 0.84
4.22 ? 2.20
0.37 ? 0.25
1.45 ? 0.61
4.55 ? 2.04
8.12 ? 3.85
1Mean relative expression of tissue factor as calculated by subtrac-
tion of the geometrical mean fluorescence intensity of the isotype-
control IgG (MOPC-21) from the geometrical mean fluorescence in-
tensity detected by the tissue factor-specific antibody TF 9-6B4. The
mean ? SD of 5–6 experiments are shown.
FIGURE 3 – Effect of the PI3-kinase-specific inhibitor wortmannin
on the activation of mitogen- and stress-activated protein kinases.
HUVECs starved in low-serum containing medium for 4 hr were
pre-incubated with 100 nM wortmannin for one additional hour. Cells
were stimulated with either 500 pM TNF for 20 min, 1 nM VEGF for
5 min or a combination of both. Cells were harvested and lysates were
subjected to Western blot analysis. The blot was incubated with
phospho-specific antibodies directed against the activated form of
p44/42 MAP kinase, p38 MAP kinase or JNK. A pan-specific p44/42
MAP kinase antibody was used to determine overall protein loading.
The membrane was stripped before reprobing.
MATSCHURAT ET AL.
However, the pro-apoptotic effect of wortmannin can be rescued
by the application of the survival factor VEGF in vitro (data not
shown). There is evidence that a higher apoptosis rate correlates
with increased tissue factor activity and a procoagulant state of
endothelial cells.51,52However, our in vivo results clearly show
that fibrin deposits are already formed as early as 3 hr after
stimulation with TNF ? wortmannin (Fig. 4m), at a timepoint
much too early for apoptosis to contribute to enhanced tissue factor
expression. In addition, the endothelial cell layer of the affected
vessels apparently remains intact, as judged by PECAM-staining
of the vessels (Fig. 4n).
Having demonstrated that the addition of wortmannin to TNF-
and VEGF-stimulated HUVECs leads to a strong increase in tissue
factor expression in vitro, we investigated the influence of wort-
mannin in combination with TNF on tumor blood vessels in vivo.
Intraperitoneal injection of 1 ?g TNF ? wortmannin into mice
bearing meth A tumors led to the development of extensive bleed-
ings within the tumors, accompanied by prominent intratumoral
necrosis formation within 24 hr (Fig. 4). In contrast, hardly any
necroses were detectable in tumors of mice treated with either
wortmannin or 1 ?g TNF only. Injection of the higher dose of 5 ?g
TNF led to a significant increase in necrosis formation compared
to the application of 1 ?g TNF. However, the mean percentage of
necrotic tissue in the tumors treated with 1 ?g TNF ? wortmannin
(22%) was still clearly higher than in the tumors of mice treated
with 5 ?g TNF (9%). Although this difference was not statistically
FIGURE 4 – Enhanced development of intratumoral hemorrhages and necroses and intravascular fibrin deposition in TNF-treated mice by
wortmannin. (a–h) Meth A tumor-bearing mice were injected intraperitoneally with solvent only (a?b), 0.4 mg/kg body weight wortmannin
(c?d), 5 ?g TNF (e?f) or 1 ?g TNF ? 0.4 mg/kg body weight wortmannin (g?h) 24 hr prior to excision of the tumors. Photographs of the
tumors were taken immediately before harvesting the tumors (a,c,e,g). Arrows indicate intratumoral hemorrhages. Size markers correspond to
2 mm. Cryosections (5 ?m) of the tumors were stained with hematoxylin/eosin (b,d,f,h). Necroses are indicated by arrowheads. Size markers
correspond to 80 ?m. (i) Morphometric analysis of whole tumor tissue of mice treated with solvent only (n?6), 0.4 mg/kg body weight
wortmannin (n?6), 1 ?g TNF (n?3), 5 ?g TNF (n?6) or 1 ?g TNF ? 0.4 mg/kg body weight wortmannin (n?6). Results are depicted in a
“Box-blot”-diagram. The bar within the boxes represents the median. Upper and lower end of the boxes indicate the 75-percentile and
25-percentile, respectively. The 90-percentile and 10-percentile are indicated as error bars. Single events outside this range are represented by
open circles. Asterisks indicate significant differences. (k–n) Detection of fibrin deposits within tumor blood vessels. Mice were treated with
either solvent (k?l) or 1 ?g TNF ? 0.4 mg/kg body weight wortmannin (m?n) for 3 hr. Double-staining of tumor sections (5 ?m) was
performed with a FITC-labeled antibody recognizing fibrin (k?m) and with a CD31-specific antibody detected by a Cy 3-conjugated secondary
antibody (l?n) to specifically identify blood vessels. White arrows indicate fibrin deposits within blood vessels of the tumor treated with 1 ?g
TNF ? 0.4 mg/kg body weight wortmannin (m). Size markers correspond to 20 ?m.
PI3-KINASE IN TNF-INDUCED TUMOR NECROSIS
significant, it indicates that the treatment of tumors with a low
concentration of TNF in combination with wortmannin is at least
as potent in inducing necroses as applying a several-fold increased
concentration of TNF. Furthermore, we observed that the total
number of tumors responding to TNF was higher if wortmannin
was co-administered compared to treatment with TNF alone. The
development of intratumoral hemorrhages and necroses in TNF-
treated meth A tumors can be attributed to the expression of
endothelial tissue factor followed by the deposition of fibrin within
the tumor blood vessels.14Zhang et al.10provided direct evidence
that endothelial tissue factor expression is required for the forma-
tion of intravascular fibrin clots. Transfection of tumor endothelial
cells with anti-sense tissue factor-cDNA and subsequent intrave-
nous application of TNF did not result in intravascular fibrin
deposits in the tumors of these animals. In confirmation of these
previous findings, we could show a strong fibrin-specific staining
of tumor blood vessels in mice treated with TNF ? wortmannin in
contrast to control tumors. Several other studies provide evidence
that the enhanced expression of tissue factor in tumor blood
vessels is a suitable strategy for tumor therapy. The extracellular
domain of tissue factor, which mediates the procoagulant activity,
was either targeted to experimentally induced11or to naturally
occurring markers on tumor endothelial cells.12,13This strategy led
to thrombus formation and, in line with our own findings, to
intratumoral necrosis formation that was very prominent after 24
hr of treatment.12Significant tumor regression was observed be-
tween 8 and 21 days after onset of treatment without apparent side
effects. In 30–40% of the mice bearing solid tumors even com-
plete tumor regression was reported,11,13which lasted 4 months or
more.11A potential drawback of these strategies can be envisioned
in targeting the rather bulky constructs to their binding partners on
the tumor endothelium. Further complications arise from the fact
that the target molecules may not be ubiquitously expressed on the
tumor endothelial cells, as it was observed for VCAM-1.12There-
fore, the here proposed concept to induce the endogenous expres-
sion of tissue factor in the tumor endothelium may be considered.
The specificity of our approach is derived from the exogenous
production of VEGF by tumor cells, which allows VEGF, TNF
and wortmannin to act synergistically on the induction of tissue
factor solely on tumor endothelial cells. Of note, we did not
observe any thrombus formation elsewhere in the mice, e.g., in the
kidney (data not shown), indicating that no systemic complications
have occurred. However, other side effects of TNF such as reduc-
tion in cardiac output were not addressed.
In conclusion, the co-application of wortmannin allows to re-
duce the dose of TNF administered and at the same time reinforces
the endothelial cell-based destruction of the tumor tissue. There-
fore, a TNF-based anti-tumor therapy circumventing the systemic
toxic side effects of TNF can be envisioned as an attractive
strategy of anti-cancer therapy.
We are grateful to D. Ma ¨nnel (Regensburg, Germany) for pro-
viding both the murine fibrosarcoma cells (BFS-1 cells) and re-
combinant human TNF ? for in vivo experiments. We would also
like to thank W. Ruf (Scripps Institute, LaJolla, CA) for the kind
donation of the tissue factor monoclonal antibody TF9-6B4 and
Cephalon, Inc. for the generous gift of CEP-11004. The skillful
support in the morphometric analysis of tumor tissue by A. Gau-
mann is gratefully acknowledged. We are thankful to M. Raper for
critically reading the article.
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